Risk assessment of substances that are both genotoxic and carcinogenic report of an International Conference organized by EFSA and WHO with support of ILSI Europe.

The European Food Safety Authority (EFSA) and the World Health Organization (WHO), with the support of the International Life Sciences Institute, European Branch (ILSI Europe), organized an international conference on 16-18 November 2005 to discuss how regulatory and advisory bodies evaluate the potential risks of the presence in food of substances that are both genotoxic and carcinogenic. The objectives of the conference were to discuss the possible approaches for risk assessment of such substances, how the approaches may be interpreted and whether they meet the needs of risk managers. ALARA (as low as reasonably achievable) provides advice based solely on hazard identification and does not take into account either potency or human exposure. The use of quantitative low-dose extrapolation of dose-response data from an animal bioassay raises numerous scientific uncertainties related to the selection of mathematical models and extrapolation down to levels of human exposure. There was consensus that the margin of exposure (MOE) was the preferred approach because it is based on the available animal dose-response data, without extrapolation, and on human exposures. The MOE can be used for prioritisation of risk management actions but the conference recognised that it is difficult to interpret it in terms of health risk.

Metabolic activation of promutagens, detectable in Ames' Salmonella assay, by 5000 X g supernatant of rat ventral prostate.

Induction of aryl hydrocarbon hydroxylase (AHH) and 7-ethoxyresorufin-O-deethylase (7-EOD) activities as well as of benzo[a]pyrene (BP) metabolite formation in rat prostatic microsomes has been demonstrated after treatment with beta-naphthoflavone (BNF). The capacity to convert promutagenic compounds to ultimate mutagenic metabolites in the Ames' Salmonella assay by 5000 X g supernatant of rat ventral prostate was investigated. Male rats were treated with BNF, polychlorinated biphenyls (PCB; Arochlor 1254), phenobarbital (PB) and the vehicle, corn oil. PCB or BNF pretreatment increased the AHH- and 7-EOD activities 100-200-fold in the rat prostate 5000 X g supernatant (S-5 fraction). Epoxide hydrolase (EH) and glutathione-S-transferase (GST) activities were not affected while UDP-glucuronosyltransferase (UDP-GT) was increased 2.2- and 2.5-fold by PCB and BNF, respectively. PB did not significantly affect any of the enzyme activities measured. A dose-dependent increase in mutagenic response versus amount of 5000 X g supernatant and promutagen (aflatoxin B1 (AFB), 2-aminofluorene (2-AF), BP) was observed. The most pronounced activation was obtained with S-5 fraction from BNF- or PCB-treated rats. The great sensitivity of prostatic AHH to certain inducers and the capacity of the prostate to produce mutagenic metabolites might be of importance for initiation of prostatic cancer by environmental factors.

Retinol (vitamin A) as an inhibitor of the mutagenicity of aflatoxin B.

Vitamin A (retinol) was shown to inhibit the mutagenic activity of aflatoxin B1 (AFB1), when added to the Ames Salmonella/mammalian microsome assay. The inhibition was dose-dependent and not caused by a direct toxic effect on the test bacteria. On the other hand the mutagenicity of diepoxybutane, a mutagen not requiring metabolic activation, was not affected by retinol, indicating that the vitamin does not act as a general scavenger of reactive compounds. The mutagenicity of AFB1 depends on the balance between formation and breakdown of aflatoxin 2,3-epoxide, the presumed ultimate mutagenic/carcinogenic metabolite. Inhibition of AFB1 mutagenicity could thus result from decreased formation or increased breakdown of the 2,3-epoxide.

Retinoids as inhibitors of ortho-aminoazotoluene-induced mutagenesis in the Salmonella/liver microsome test.

Earlier, we reported that vitamin A (retinol), in vitro, exhibits a strong inhibitory effect on the mutagenicity of aflatoxin B1 in the Ames Salmonella/microsome test. This paper reports the same inhibitory effect on the mutagenicity of an aminoazo dye, ortho-aminoazotoluene. Furthermore, the inhibitory effect was exerted by retinol esters such as retinol acetate and retinol palmitate, the latter being the physiological storage form of vitamin A. The inhibition is interpreted as an effect on the mixed-function oxidases that convert ortho-aminoazotoluene to its ultimate mutagenic form.

Genotoxic activity of 1,3-butadiene and nitrogen dioxide and their photochemical reaction products in Drosophila and in the mouse bone marrow micronucleus assay.

The genotoxic activity of a photochemical reaction mixture of 1,3-butadiene and nitrogen dioxide was investigated in vivo in the mouse bone marrow micronucleus assay and the somatic mutation and recombination test in Drosophila (the wing spot test). Butadiene alone was not mutagenic in Drosophila, but induced micronuclei in mice at 10 ppm after 23 h of exposure. Nitrogen dioxide was not genotoxic in either test system. The photochemical reaction products were toxic but probably not mutagenic in Drosophila and not genotoxic in mouse bone marrow. The in vivo results do not confirm earlier in vitro results that demonstrated a strong direct-acting mutagenic activity of the photochemical products in Salmonella.

Retinol (vitamin A) inhibits the mutagenicity of o-aminoazotoluene activated by liver microsomes from several species in the Ames test.

Retinol (vitamin A) has earlier been shown to inhibit the mutagenicity of o-aminoazotoluene (OAAT) in the Salmonella/microsome assay when OAAT is activated with S9 from Sprague-Dawley rats. The results presented in this paper confirm this and also show that S9 from mice, hamsters and gerbils activates OAAT to mutagenic metabolites detected by Salmonella typhimurium TA100. However, S9 from rabbits is inactive. The S9 fraction from rabbits also shows a low aryl hydrocarbon hydroxylase (AHH) activity. The AHH activity or protein content of the microsomal fraction cannot be used to predict the activating capacity of S9 from the other species. Retinol, added in vitro, inhibits the mutagenic effect of OAAT activated by mouse, gerbil or hamster S9. The strongest inhibition is observed with hamster S9 while the inhibition of mouse and gerbil S9 is lower but still higher than in the rat.

The influence of pH on the effects of 2,4-D (2,4-dichlorophenoxyacetic acid, Na salt) on Saccharomyces cerevisiae and Salmonella typhimurium.

The genetic effects of 2,4-D (2,4-dichlorophenoxyacetic acid, Na salt) have been investigated in cells of the yeast Saccharomyces cerevisiae and of the bacterium Salmonella typhimurium in experiments in vitro and in vivo. Experiments in vitro showed that the killing of both yeast and bacteria is dependent on the pH in the treatment solution of 2,4-D. A dose-dependent increase of the frequency of mitotic gene conversion and mitotic recombination in yeast was observed at pH 4.50 and 4.30. In experiments in vitro with two strains of Salmonella no significant increase of the number of revertants to prototrophy was obtained. The positive correlation between survival of cells and dissociation of 2,4-D in the pH region 2.8-5.0 indicates that the cells are unable to take up dissociated 2,4-D. Therefore the survival is high at a high pH when most 2,4-D is in dissociated form, and the survival is low at a relatively low pH when more of the 2,4-D is in its undissociated form. No genetic effects were induced by oral administration of tolerable doses of 2,4-D in host-mediated assays using mice as hosts and yeast or Salmonella as indicator cells.

Effects of vitamin A on cyclophosphamide mutagenicity in vitro (Ames test) and in vivo (mouse micronucleus test).

The effect of vitamin A on cyclophosphamide mutagenicity was measured both in vitro and in vivo. In the Ames test in Salmonella typhimurium TA1535 with mouse-liver S-9 mix, the addition of retinol, retinyl acetate or retinyl palmitate caused a dose-dependent inhibition of cyclophosphamide mutagenicity. In the micronucleus test in male NMRI mice fed low, normal or high levels of vitamin A, the induction of micronuclei in bone marrow by an ip dose of cyclophosphamide was unaffected by vitamin A status. Thus, this study provides no evidence that activation of a procarcinogen in the liver or bone marrow of mice can be modified by vitamin A. One of the possible reasons for the observed absence of inhibition by vitamin A in vivo may be a lack of correlation between the oral dose of retinoid and the resulting level of vitamin A in the bone marrow. The difference between results in vitro and in vivo may also have been due to a difference in the availability and potency of added vitamin A in vitro compared with the forms absorbed and stored in vivo.

Inhibition of protein pyrolysate mutagenicity by retinol (vitamin A).

The mutagenicity in the Ames Salmonella-microsome test of four protein pyrolysate products, formed during the cooking of meat, (Trp-P-1, Trp-P-2, Glu-P-1 and Glu-P-2) was found to be inhibited by the addition of vitamin A in vitro in the form of retinol. The effect is interpreted as an inhibition of the metabolic activation of the mutagens to their respective ultimate mutagenic forms since retinol has been shown to have no effect on the survival of the Salmonella cells, no effect on directly acting mutagens and no effect on the formation of NADPH in the test system. The results demonstrate the need for an increased understanding of the interaction of dietary components in evaluating mutagenic/carcinogenic risks from processed food.

Long-term dietary exposure to lead in young European children: comparing a pan-European approach with a national exposure assessment.

Long-term dietary exposures to lead in young children were calculated by combining food consumption data of 11 European countries categorised using harmonised broad food categories with occurrence data on lead from different Member States (pan-European approach). The results of the assessment in children living in the Netherlands were compared with a long-term lead intake assessment in the same group using Dutch lead concentration data and linking the consumption and concentration data at the highest possible level of detail. Exposures obtained with the pan-European approach were higher than the national exposure calculations. For both assessments cereals contributed most to the exposure. The lower dietary exposure in the national study was due to the use of lower lead concentrations and a more optimal linkage of food consumption and concentration data. When a pan-European approach, using a harmonised food categorisation system and "European" concentration data, results in a possible health risk related to the intake of an environmental chemical for a certain country, it is advisable to refine this assessment, as part of a tiered approach, using national occurrence data, including an optimised linkage between foods analysed and consumed for that country. In the case of lack of occurrence data, these data can be supplemented with data from the "European" concentration database or by generating additional concentration data at country level.

Harmonisation of food categorisation systems for dietary exposure assessments among European children.

Within the European project called EXPOCHI (Individual Food Consumption Data and Exposure Assessment Studies for Children), 14 different European individual food consumption databases of children were used to conduct harmonised dietary exposure assessments for lead, chromium, selenium and food colours. For this, two food categorisation systems were developed to classify the food consumption data in such a way that these could be linked to occurrence data of the considered compounds. One system served for the exposure calculations of lead, chromium and selenium. The second system was developed for the exposure assessment of food colours. The food categories defined for the lead, chromium and selenium exposure calculations were used as a basis for the food colour categorisation, with adaptations to optimise the linkage with the food colour occurrence data. With this work, an initial impetus was given to make user-friendly food categorisation systems for contaminants and food colours applicable on a pan-European level. However, a set of difficulties were encountered in creating a common food categorisation system for 14 individual food consumption databases that differ in the type and number of foods coded and in level of detail provided about the consumed foods. The work done and the problems encountered in this project can be of interest for future projects in which food consumption data will be collected on a pan-European level and used for common exposure assessments.

Probabilistic modelling of exposure doses and implications for health risk characterization: glycoalkaloids from potatoes.

Potatoes are a source of glycoalkaloids (GAs) represented primarily by alpha-solanine and alpha-chaconine (about 95%). Content of GAs in tubers is usually 10-100 mg/kg and maximum levels do not exceed 200 mg/kg. GAs can be hazardous for human health. Poisoning involve gastrointestinal ailments and neurological symptoms. A single intake of >1-3 mg/kg b.w. is considered a critical effect dose (CED). Probabilistic modelling of acute and chronic (usual) exposure to GAs was performed in the Czech Republic, Sweden and The Netherlands. National databases on individual consumption of foods, data on concentration of GAs in tubers (439 Czech and Swedish results) and processing factors were used for modelling. Results concluded that potatoes currently available at the European market may lead to acute intakes >1 mg GAs/kg b.w./day for upper tail of the intake distribution (0.01% of population) in all three countries. 50 mg GAs/kg raw unpeeled tubers ensures that at least 99.99% of the population does not exceed the CED. Estimated chronic (usual) intake in participating countries was 0.25, 0.29 and 0.56 mg/kg b.w./day (97.5% upper confidence limit). It remains unclear if the incidence of GAs poisoning is underreported or if assumptions are the worst case for extremely sensitive persons.

Hepatic and extrahepatic bioactivation and GSH conjugation of aflatoxin B1 in sheep.

Whole-body autoradiography of [3H]aflatoxin B1 ([3H]AFB1) in lamb showed a localization of bound labelling, in addition to the liver, in the nasal olfactory and respiratory mucosa, in the mucosa of the nasopharynx, pharynx, oesophagus, larynx, trachea, bronchi and bronchioles and in the palpebral and bulbar conjunctiva. Microautoradiography revealed that the bound material was confined to specific cell types in extrahepatic tissues. Whole-body autoradiography also showed a labelling of pigmented tissues (such as the eye melanin), which can be ascribed to a melanin affinity of AFB1. In vivo experiments, performed with microsomal preparations of tissues from ewe and lamb showed that several of the extrahepatic tissues were more efficient than the liver in forming DNA-bound AFB1 metabolites. The nasal olfactory mucosa was by far the most effective tissue in this respect. AFB1 induced a high number of gene mutations in Salmonella typhimurium TA100 when incubated with supernatant preparations (9000 g) of the nasal olfactory mucosa, whereas incubations with preparations of the liver resulted in a lower effect. It has been reported that AFB1 can induce nasal tumours in sheep. When microsomal preparations of various tissues were incubated in the presence of reduced glutathione (GSH), but without any addition of cytosolic glutathione-S-transferase (GST), a drastic decrease in the AFB1-DNA binding was seen. Analyses of the water-soluble metabolites formed in the microsomal incubations supplemented with GSH showed fluorescent and ninhydrin-positive metabolites that were not present in the absence of GSH. These results indicate that sheep tissues have intrinsic microsomal GST or cytosolic GSTs associated with the microsomal fraction with a high capacity to catalyse the conjugation of bioactivated AFB1 to GSH. The results of the present study show that several extrahepatic tissues of sheep have a potent capacity to bioactivate AFB1 and also a high capacity to GSH conjugate the bioactivated AFB1.

Retinol (vitamin A) as a modifier of 2-aminofluorene and 2-acetyl-aminofluorene mutagenesis in the Salmonella/microsome assay.

Vitamin A (retinol)has been demonstrated to modify the mutagenic activity of the aromatic amines, 2-aminofluorene (2AF) and 2-acetylaminofluorene (2AAF) when added to the Ames Salmonella/mammalian microsome assay. Low amounts of retinol(2-20 micrograms/plate) increased the mutagenicity of both 2AF and 2AAF. At higher doses (50-150 micrograms/plates) the mutagenicity of 2AAF remained unchanged while the mutagenicity of 2AF gradually decreased. The present data do not support the hypothesis that retinol generally acts as an inhibitor of in vitro metabolic activation of procarcinogens.

Bioactivation of aflatoxin B1 in the bovine olfactory mucosa: DNA binding, mutagenicity and induction of sister chromatid exchanges.

Nasal olfactory tumours occur in cattle in relatively high frequencies in several developing countries. Since affected animals sometimes show signs of severe aflatoxicosis, a role of aflatoxin B1 (AFB1) in tumorigenesis can be proposed. The results of the present study show that microsomal preparations of the bovine olfactory mucosa have a much higher ability than liver microsomes to induce covalent binding of AFB1 to calf thymus DNA and to microsomal proteins. The major DNA adduct formed was 8,9-dihydro-8-(N7-guanyl)-9-hydroxy-aflatoxin B1. Incubations of microsomal preparations of the bovine nasal olfactory mucosa with glutathione (GSH) and cytosolic fractions of the nasal mucosa resulted in decreased AFB1 DNA binding. A more pronounced decrease was observed when cytosolic fractions of mouse liver were added to the incubations. Mouse liver is known to contain a glutathione-S-transferase with a high ability to scavenge the reactive AFB1-epoxide via conjugation to GSH. Our results indicate that AFB1-GSH conjugation occurs less efficiently in the bovine nasal olfactory mucosa than in the mouse liver. Supernatant preparations (9000 g) of the bovine nasal olfactory mucosa incubated with AFB1 were shown to have the capacity to induce a strong genotoxic response both as regards induction of gene mutations in Salmonella typhimurium TA100 and the induction of sister chromatid exchanges in Chinese hamster ovary cells. Preparations of the bovine liver (9000 g) has a much lower ability to induce these effects. The results of the present study show that the bovine nasal olfactory mucosa has a high AFB1-bioactivating capacity, which can be related to the potent DNA damaging and mutagenic effects observed. It is considered that our results support the assumption that AFB1 plays a role in the aetiology of nasal tumours in cattle.

Effects of vitamin A on the metabolism and mutagenicity of 2AAF in vitro and on the covalent binding to rat liver DNA/RNA in vivo.

Vitamin A has been shown to affect the in vitro metabolism of 2AAF. At low concentrations of retinol or retinyl palmitate, a decreased production of ring-hydroxylated as well as deacetylated and N-hydroxylated metabolites was observed, measured by high performance liquid chromatography. The increased mutagenicity of 2AAF observed after addition of vitamin A in the Ames test cannot therefore be explained as a result of stimulated N-hydroxylation. However, the addition of retinol was found to enhance the mutagenicity of the metabolite N-OH-2AAF in the presence of an S-9 fraction of rat liver homogenate. No differences with regard to the covalent binding of 2AAF or its metabolites to rat liver DNA/RNA in vivo could be demonstrated in animals fed diets with normal or high vitamin A content.

3-Aminobenzamide: effects on cytochrome P450-dependent metabolism of chemicals and on the toxicity of dichlobenil in the olfactory mucosa.

Treatment with 3-aminobenzamide, known as an inhibitor of poly(ADP-ribose)polymerease, decreased the toxicity and covalent binding of the herbicide dichlobenil (2,6-dichlorobenzonitrile; 12 mg/kg; i.p.) in the mouse olfactory mucosa. In vitro studies showed that 3-aminobenzamide markedly reduced the NADPH-dependent covalent binding of [14C]dichlobenil and the hydroxylation of p-nitrophenol which have previously been suggested to be mediated by a common form of cytochrome P450 (P450) in rat olfactory microsomes (Eriksson and Brittebo, Chem.-Biol. Interact. 94,183-196, 1995). Furthermore, 3-aminobenzamide markedly reduced the P450-dependent metabolic activation of [3H]NNK (4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone) in rat olfactory microsomes and slightly decreased the P450 2B1-dependent pentoxyresorufindealkylase activity in liver microsomes of phenobarbital-treated rats. The present results suggest that 3-aminobenzamide is also an inhibitor of P450 and that the lack of toxicity of dichlobenil in the olfactory mucosa of 3-aminobenzamide-treated mice is related to a decreased metabolic activation of dichlobenil at this site. Further experiments showed that there was no evidence for a binding of [14C]dichlobenil metabolites to calf thymus DNA or a formation of mutagenic dichlobenil metabolites in Ames' Salmonella assay when dichlobenil was incubated in the presence of homogenates of the olfactory mucosa. Finally, analysis of proteins from olfactory microsomes incubated with [14C]dichlobenil using SDS-PAGE/fluorography revealed a binding of metabolites to all major proteins. Addition of glutathione or the P450-inhibitor metyrapone prevented the binding, suggesting the formation of relatively stable electrophilic products which can leave the activating enzyme and then unselectively bind to the major olfactory microsomal proteins.

Effect of o,p'-DDD on the in vivo incorporation of 3H-thymidine into DNA: evidence for induced cell proliferation in the mouse lung.

The effects of o,p'-DDD on the DNA synthesis in the C57Bl mouse lung and liver were studied. As determined by 3H-thymidine incorporation into DNA, a selective increase in the lung DNA synthesis (+59%) was observed 2 days after a single intraperitoneal injection of 100 mg/kg o,p'-DDD. Microautoradiography showed that the incorporated 3H-thymidine was confined to a restricted number of heavily labelled cells, presumably proliferating type II cells. At the most, a 9 times higher rate of cell proliferation was observed in the lung 4 days after an intraperitoneal injection of 500 mg/kg o,p'-DDD. Using mouse lung or liver S-9 as activating system, no mutagenic activity of o,p'-DDD was detected in the Ames test. The induced cell proliferation may indicate a tissue-selective promoter activity of o,p'-DDD in the mouse lung.